The present invention relates to a skin autograft composite comprising human epidermal cells and usable, for example, in the treatment of burn victims. Effective and readily usable methods for treating substantial injuries to skin have long been goals of physicians and scientists alike. Extensive dermal injury is most frequently caused by burns. With severe burns the epidermal and dermal skin structures in particular areas may be so severely damaged that no epidermal cells remain to repopulate as skin. While numerous skin substitutes such as pigskin, for example, have been tried as coatings for burn wounds, none has proved satisfactorily effective.
Many efforts have been made to facilitate the regrowth of epidermal cells on areas denuded of native skin structures. Because of individual immunological characteristics, the utilization of a burn victim's own epidermal cells to create a new skin covering has been accepted as an ideal potential remedy for such skin injuries. A skin autograft comprising an epidermal cell population would constitute such a new skin covering or an initiation of the regrowth leading to a natural skin substitute. The preparation of an epidermal cell population for purposes of autograft formation may involve in vitro cell culture and possibly an extensive expansion in the number of desired cells.
Human diploid epidermal cells have been grown in culture in the presence of fibroblasts. However, proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. This requires plating epidermal cells with irradiated 3T3 (mouse) cells. Rheinwald and Green, Cell. 6, 331-334, November 1975. This technique also requires the presence of dermal components and cell culture to full differentiation. Despite these limitations, which the yield far from optimal, it has been used for grafts on burn patients.
Kitano et al. suggest that keratinocytes dispersed from epidermis grow without dermal components in a suitable culture medium (30% fetal bovine serum) and show some signs of differentiation (Biochemistry of Cutaneous Epidermal Differentiation, Ed. by Seiji etal. University Park Press, 1977, pp. 319-335). However, this technique has not solved the skin autograft problem.
Approaches to the use of epidermal cells cultured and expanded in vitro and used as skin autografts are included in U.S. Pat. Nos. 4,254,226 and 4,299,819. These references describe processes generally involving: separation of the epidermis from the dermis in samples of human skin; dissociation of the epidermis into epidermal cells; growth of the epidermal cells into a pure epidermal sheet in a tissue culture medium without dermal components and having a pH of from about 5.6 to about 5.9; and application of the resultant epidermal cell sheet to an afflicted area of a burn victim.
Certain earlier efforts generally related to objects of the present invention may be described as follows:
Knazek etal. (U.S. Pat. Nos. 3,883,393 and 4,220,725) describe a system that is to grow cells as three-dimensional structures in semi-permeable tubes. Epidermal undifferentiated basal cells do not grow as three-dimensional structures but instead as a monolayer. (Cancerous epidermal cells grow as undifferentiated three-dimensional structures; indeed this is a standard test for neoplasia). Cells growing inside a capillary tube do not constitute a suitable material for a skin graft.
Feder etal. (U.S. Pat. No. 4,087,327) describe a geometrically complex analog of the Knazek system. The same comments apply.
Verma (U.S. Pat. No. 4,296,205) describes a system for continuous growth of a limited number of cells by the continuous dialysis of the culture media. Epidermal cells in such a system would differentiate and die and are, therefore unsuitable as a graft.
Jarvis et al. (U.S. Pat. No. 4,495,288) describe a culturing system of anchorage dependent cells in suspension. Epidermal cells will not grow in suspension.
Leighton etal. (U.S. Pat. No. 4,308,351) describe a system designed for the growth of pathological and cancer cells. The cells were nurtured through a semi-permeable membraneous wall.
Oliver etal. (U.S. Pat. No. 4,399,123) describe fibrous tissue (dermis) preparations for transplantation. Epidermis is not a fibrous tissue. The present invention involves an intact cell layer of epidermal cells.
Bell (U.S. Pat. Nos. 4,485,096 and 4,539,716) and Yauas et al. (U.S. Pat. No. 4,060,081) describe synthetic skin substitutes. These may be alternatives to grafting but they are unrelated to the autografts of the present invention.
Eisinger (U.S. Pat. No. 4,299,819) and Eisinger etal. (U.S. Pat. No. 4,254,226) describe fully differentiated epidermal cells in a low pH culture medium. To use these cells as grafts, they were attached to the dermal side of pig skin or a collagen sponge and then used this sticky covering to yank the cultured cells loose from their attachment to the culture vessel. In this fully differentiated system there could have been but few dividing cells (undifferentiated) altogether, and many no doubt were lost or damaged during any transfer process onto a wound.
In the usual procedures used by other laboratories, the epidermal cells are fully differentiated. They are lifted by yanking from the culture vessel or are detached enzymatically by dispase. The cells in the latter procedure shrink to about one-half their surface area. These procedures are needed in order to protect the structural integrity of the cells used for a graft.
Where an individual has had extensive skin damage, for example from burns, a graft may be immunologically rejected unless the cells being grafted are from the same individual. To expand the available epidermis, epidermal cells may be grown in culture and then used as grafts.
Technical transfer difficulties such as those involved in detaching cells from culture vessels, spreading them over the wound and providing them with appropriate dressings have impeded rapid and effective treatment of dermatological injuries. Enzymatic detachment of cultured epidermal cells from cell culture vessels, for example, has resulted in up to 75% shrinkage while mechanical transfer (picking up the cells on a rigid artificial structure and then loosening them at the wound) has resulted in substantial cell loss and damage.
The above designated and other analogous known methods of skin autografting may represent progress in the field but have not proven completely satisfactory. Problems unsolved by these earlier methods include: insufficient number of undifferentiated epidermal cells for grafting; difficulties in the removal of an epidermal sheet from a tissue culture container; shrinkage and partial destruction of the epidermal sheet; paucity of viable epidermal cells; and a lack of a sound and physiologically acceptable graft covering. The processes of the present invention permit these and other related problems to be minimized or solved.